According to the prior art, quantitative determination of glycerol in a sample which uses glycerol dehydrogenase is conducted in line with the following equation:
Glycerol+NAD.sup.+ .dbd.Dihydroxyacetone+NADH (glycerol dehydrogenase) PA1 (1) It catalyzes the following reaction: Glycerol+Electron Acceptor.dbd.Glyceraldehyde+Reduced Electron Accept PA1 (2) Optimum pH: pH 8-9 PA1 (3) pH stability: Stable at pH 7-11 PA1 (4) Optimum temperature: 20.degree.-25.degree. C. PA1 (5) Thermal stability: Stable at a temperature of 30.degree. C. or less for 10 minutes' reaction at a pH of 7.0, PA1 (6) Molecular weight: 70,000 by gel filtration and SDS polyacrylamide gel electrophoresis. PA1 (7) Contains pyrroloquinoline quinone a prosthetic group. PA1 (8) No surfactant needed for its solubilization and stabilization. PA1 (1) It catalyzes the following reaction: Glycerol+Electron Acceptor.dbd.Glyceraldehyde+Reduced Electron Acceptor PA1 (2) Optimum pH: pH 8-9 PA1 (3) pH stability: Stable at pH 7-11 PA1 (4) Optimum temperature: 20.degree.-25.degree. C. PA1 (5) Thermal stability: Stable at a temperature of 30.degree. C. or less for 10 minutes at a pH of 7.0, PA1 (6) Molecular weight: 70,000 by gel filtration and SDS polyacrylamide gel electrophoresis. PA1 (7) Contains pyrroloquinline quinone as a prosthetic group. PA1 (8) No surfactant needed for its solubilization and stabilization. PA1 (a) Morphological characteristics: PA1 (b) Cultural characteristics: PA1 (c) Physiological characteristics: -
In view of the disadvantage of utilizing the expensive coenzyme NAD.sup.+ in this process, we the inventors of the present invention decided to develop a cheap and simple method for quantitatively determining glycerol, focusing on PQQ-dependent glycerol dehydrogenase which utilizes an artificial electron acceptor. The glycerol dehydrogenase includes the one already reported which is produced by acetic acid bacterium, Gluconobacter industrious IFO 3260 (Ameyama et al., Agric. Biol. Chem., 49 1001-1010, 1985). The enzyme is, however, a cell membrane-bound type, and thus its solubilization through separation from the cells and its stabilization requires the use of a certain surfactant which may cause errors in the measurement of glycerol. Further, the enzyme is a rather highly hydrophobic membrane enzyme, and therefore is unstable in an aqueous solvent system, requiring the addition of 10% glycerol thereto as a stabilizing agent, in addition to a surfactant. These facts indicate why the enzyme cannot be used for quantitative determination of glycerol.